Extended Data Fig. 2 |. xmOVA pregnant mice show a mild, OVA-dependent expansion of OVA-specific Tregs, but Treg depletion does not alter their suppressed CD4⁺ T cell response to t-mOVA.

Treg depletion was accomplished through use of the Foxp3^DTR system, in which the gene for the diphtheria toxin receptor (DTR) is knocked into the X-linked Foxp3 locus, thus rendering Tregs sensitive to diphtheria toxin- (DT-) induced ablation³⁵. Since complete Treg ablation starting at mid-gestation is known to cause near-total pregnancy failure¹², our experiments employed Foxp3^DTR/WT female mice in which, due to random X-inactivation, ~50% of CD4⁺ T cells express a wild-type Foxp3 allele and the other ~50% express the DTR knock-in allele. DT administration thus causes a ~50% acute reduction in Treg frequencies³⁵. While this reduction is transient, it is still sufficient to induce a significant degree of fetal loss in allogeneic mating combinations¹². By contrast, partial Treg ablation in the syngeneic mating combinations employed here (C57BL/6 × C57BL/6, aside from the mOVA transgene) did not induce fetal loss. To prevent the transferred OT-II cells themselves from generating an OVA-specific Treg population, we also employed OT-II Foxp3^DTR/Y males as cell donors. All FOXP3⁺ OT-II cells from these mice are ablatable since they all express the Foxp3^DTR allele. a, Frequency of FOXP3⁺ Treg OT-II cells among total splenic OT-II cells 6 days after adoptive transfer into virgin mice or mid-gestational (E12.5–15.5) WT or Foxp3^DTR/WT mice mated as indicated. The Foxp3^DTR/WT mice received OT-II Foxp3^DTR/Y cells, and were injected daily with DT starting at E10.5, in line with previously work¹². Note that virtually none of the transferred cells in this latter group converted into Tregs. Adjusted P-values were determined by ordinary one-way ANOVA with Šídák’s multiple comparisons test applied to the four comparisons shown. ns, not significant. Data were accumulated from 5 individual experiments and all mice are shown. b, Confirmation of partial, DT-induced depletion of host FOXP3⁺ CD4⁺ cells in Foxp3^DTR/WT female mice. The frequency of FOXP3⁺ cells among total CD4⁺ lymphocytes in the spleens of virgin Foxp3^DTR/WT female mice was measured 24 h after DT administration. P-value was determined by two-tailed, unpaired t-test. Data are from 1 experiment and all mice are shown. c-f, Proliferation index (c), fold expansion (d), activation marker expression (e), and IFN-γ production (f) of CFSE-labeled Foxp3^DTR/Y OT-II cells 6 days after adoptive transfer on E12.5–15.5 into Foxp3^DTR/WT mice mated as indicated. The mice were injected daily with DT starting on E10.5, thus partially depleting endogenous Tregs and completely ablating all OT-II cells that have converted into Tregs, as described above. Some groups received i.v. adjuvants (poly(I:C)+anti-CD40 antibodies) ± c-OVA at the time of OT-II transfer. Adjusted P-values were determined by ordinary one-way ANOVA with Šídák’s multiple comparisons test. Each group was compared to the xB6 control group, and the xB6+c-OVA/Adj group was compared to the xmOVA+c-OVA/Adj group. Bars show mean±s.d. Data are from 4 independent experiments and all mice are shown.